Eur. J. Biochem. 205, 527-535 (1992) ):T FERS 1992

Structural studies on fetal mucins from human amniotic fluid Core typing of short-chain 0-linked glycans Fran~GcorgHANISCH and Jasna PETER-KATALINIC' Institute of Imrnunobiology, University Clinic of Cologne, Federal Republic of Germany Institute of Physiological Chemistry, Universily of Bonn, Federal Republic of Germany (Receivcd December 9, 1991) - EJB 91 1647

Mucins in human amniotic fluid are represented by two distinct molecular species, FM-1 and FM-2, with apparent molecular masses of 700 and 570 kDa, respectively, in SDS/polyacrylamide gradient gels. FM-1 and FM-2 were isolated by preparative SDSjPAGE to apparent homogeneity and subjectcd to structural studies on their carbohydrate portions. The carbohydrate compositions of the mucin species differed only marginally and exhibited significant amounts of mannose. 0-linked core-region glycans on human amniotic mucin-derived pronase-stable glycopeptides were analyzed after reductive p-elimination and purification on HPLC by a combination of methylation analysis, electron-impact mass spectrometry of permcthylated oligosaccharide alditols and Fastatom-bombardment mass spectrometry of acetylated or methylated alditols (positive-ion mode) or alditol-derived neoglycolipids (negative-ion TLC-MS). The primary structures of major monosaccharides to tetrasaccharides have been established which exhibit at their reducing termini core 1, core 2 and core 3 sequences, as follows. GalN Ac-ol Galfi(1 -3)GalNAc-ol

Fuca(l-2)Galfi(1-3)GalNAc-ol Galp( l-3)GlcNAc~(1-3)GalNAc-ol

Galj?(1-4)GlcNAcfi(1-3)GalNAc-ol Galp( 1-3)[GlcNAc~(1-6)]GalNAc-o1 Gal/l( 1-4)[F~~a(l-3)]GlcNAcfi( 1-3)GalNAc-ol Galp( 1-3)[Galp(I -4)GlcNAcp( 1-6)]GalNAc-ol

Mucins from human amniotic fluid, previously designated as amnion mucoids, were first isolated by Lambotte and Uhlenbruck in 1966 [I] and characterized as lacking blood group expression. Amniotic mucins are thought to be derived, at least in part, from fetal secretions and should, accordingly, be related to meconium glycoproteins [2]. Structural and immunochemica1 evidence from previous studies suggested that amniotic mucins express large amounts of oncofetal carbohydrate antigens on sialylated glycans belonging to the Lex.Y/Le"sb family [3, 41. Moreover, hybridoma-defined carbohydrate epitopes Correspondtwcc. t o F.-G. Hanisch, Institute of Immunobiology, University Clinic of Cologne, Kerpener Strasse 15, W-5000 Cologne 41, Federal Republic of Germany Ab/~rtviutions. Fuc, fucose; GalNAc-ol, N-acetylgalactosaminitol; Hex, hexose; HexNAc, N-acetylhcxosamine; FAB-MS, Fdst-atom-bornbardmcnt mass spectrometry; El-MS electron-impact mass spcctrometry. Enzymes. p-N-Acetylglucosaminidasc from jack beans (EC 3.2.1.30); B-galactosidase (EC 3.2.1.23) and a-galactosidase (EC 3.2.1.22) from Escherichiu c n l i ; a+fucosidase from bovine kidney (EC 3.2.1.51).

on human amniotic mucins have recently been demonstrated to be associated with carcinomas of the distal colon [S]. The present investigation was performed to characterize the structural attributes of fetal mucin carbohydrates and to indicate the possible relationship between mucins from amniotic fluid and meconium. 0-linked glycans on meconium glycoproteins have been extensively analyzed [6- 81 and reported to comprise a series of oligosaccharides that are expressed also on mucins from human colonic adenocarcinoma [9] or from normal colonic tissue [lo]. Among these, the core-region sequences of 0-linked glycans exhibit most characteristic structural features which, according to present knowledge, show some restriction to this organ in humans. In particular, core 3 sequences, GlcNAcp(1-3)GalNAc (see also Table 6), have been found on human colonic mucins [lo] and meconium glycoproteins [7], while core S sequences, GalNAca( 1-3)GalNAc, in human tissues seem to be restricted to fetal and carcinoma-associated mucins being expressed in meconium [7] and colonic adenocarcinoma [9]. Other core-types like core 1, GalB(13)GalNAc, or, in particular, core 2, GalP(1-3)[GlcNAcP(1 6)]GalNAc, show a rather broad species and organ distri-

528 Universitatsklinikum Steglitz, Berlin-Steglitz, FRG), A005. bution, while core 4, GlcNAc~~(1-3)[GlcNAc~(1-6)]GalNAc, B006, Le"O1, LebOl (Bio-Carb, Lund, Sweden). again displays a more restricted pattern of expression. A variety of methods has been used to establish the core types of O-linked glycans, among these electron-impact mass spectrometry (EI-MS), which allows assignment of glycan Immunochemical analyses branches to substitution positions of the core GalNAc by the Immunochemical analyses of antibody binding to pronaseformation of structurally relevant alditol fragments. Most stable glycopeptides or to mucins FM-1 and FM-2 were recently a method has been introduced by Stoll et al. [ I l l performed by drying 50-pl aliquots of 0.01 mg antigen/ml allowing similar structural assignments by thin-layer chromatography negative-ion mass spectrometry (TLC-MS) carbonate buffer, pH 9.3 in flat-bottom polystyrene wells at of native neoglycolipids, which are derived from oxidative 37°C. After blocking the residual surface activity with 5 % cleavage of the C4-C5 bond in the terminal alditol. In con- bovine serum albumin in 20 mM phosphate, 0.15 M NaCI, junction with fast-atom-bombardment mass spectrometry pH 7.2 (P,/NaCI) for 3 h at 37'C, the wells were incubated (FAB-MS) (pseudo-molecular ions), methylation analysis and with 50-pl aliquots of antibody dilutions in 0.5% albumin in C r 0 3 oxidation, these methods are suitable for the complete Pi/NaC1( 5 - 10 pg/ml) overnight at 4°C. Bound antibody was structural characterization of mixtures of glycan alditols, in detected with 2259 anti-mouse immunoglobulin (Dako, particular, with regard to the analysis of minor components Hamburg, FRG) and monoclonal anti-(alkaline-phosphatase)/alkaline-phosphatase complex in a double-sandwich asexhibiting alternative core types and branching patterns. say using 4-nitrophenyl phosphate as substrate in diethanolamine buffer (1 mg/ml) containing 0.5 mM MgCI2. The enzymatic product was colorirnetrically analyzed at 405 nm in a MATERIALS AND METHODS Dynatech microplate reader (MR 5000). Preparation of pronase-stable, high-molecular-mass glycopeptides Samples of human amniotic fluid were collected on delivery at the University Clinic of Gynecology and Obstetrics by specially instructed nurses. A total of 2.5 1 pooled samples were thawed and centrifuged at 25000 g for 30 min (4°C). The clear, cell-free supernatant was digested with 100 mg pronase P (StreptonzyccJ.9 grisrus protease, Serva, Heidelberg, FRG) after preincubation of the enzyme for 1 h at 37°C in 0.05 M Tris, 0.15 M NaCI, 5 mM CaCI,, pH 7.8. Proteolysis was performed at pH 7.8 under a layer of toluene for 40 h, with further addition of fresh enzyme after 24 h and readjustment of the pH to 7.8. Urea was added to the reaction mixture (6 M ) and the pH was adjusted to 12 with 1 M NaOH prior to extraction of pronase-stable glycopeptides with an equal volume of phenol at 65°C (10 min). Separation of the phases was achieved by addition of 1 vol. water and centrifugation at 5000 g (1 h). The aqueous phase was extensively dialysed against water with several changes for a total period of three days. After freeze-drying 1 g glycopeptides (total yield 2.75 g) was applied onto a column (2.5 x 100 cm) of Bio-Gel P 30 (100 - 200 mesh) equilibrated in 0.05 M pyridine/acetate, pH 5.0, and fractionated at a flow rate of 24 ml/h. The eluate was registered spectrophotometrically at 280 nm or colorimetrically by the phenol/sulfuric acid or thiobarbituric acid methods for the analysis of hexose or sialic acid, respectively. Glycopeptides eluting in the void volume were freeze-dried (500 mg) and analyzed for their carbohydrate content (80% by mass), their monosaccharide compositions (mannose amounting to approximately 4% of total neutral carbohydrates) and their binding activities for various monoclonal antibodies with carbohydrate specificities, including the hybridoma antibodies CSLEXI (Dr Terasaki, UCLA School of Medicine, University of California, Los Angeles, USA), B72.3, SP21 (Biogenex Laboratories, San Ramon, CA, USA), NS19-9 (Dr Del Villano, Centocor, Malvern, PA, USA), LeuMl (Beckton-Dickinson, Mountain View, CA, USA), A46B/B10 (Dr Karsten, Zentralinstitut fur Molekularbiologie, Berlin-Buch, FRG), 49H8 (Dr Longenecker, Department of Immunology, University of Alberta, Edmonton, Canada), 12-4LE (Dr Bara, Institute des Recherches Scientifiques sur le Cancer, Villejuif Cedex, France), AM-3 (Dr Hanski,

Analytical and preparative gel electrophoresis Amniotic glycoproteins (20 mg) obtained by phenol cxtraction of 114ml amniotic fluid [4] were separated by gel electrophoresis in 3 - 12% polyacrylamide gradient gels (1.5 mm thick) in the presence of 0.1% SDS at 45 mA using a Bio-Rad apparatus. A 5O-pl sample was introduced/slot containing 500 pg glycoprotein, which had been preincubated in 1% SDS, 5% 2-mercaptoethanol for 10 min at 90 C. Gels were fixed and stained with periodic acid/Schiff reagent or cut into 5-mm strips, followed by electrophoretical elution in a Bio-Rad electro-eluter (model 422). Mucin-containing fractions FM-1 and FM-2 were extensively dialyzed against 40% aqueous methanol, freed from methanol by vacuum evaporation and freeze-dried. The dry masses were 3.7 mg (FM1) or 3.2 mg (FM-2), respectively, corresponding to 0.9 mg (FM-1) or 0.7 mg (FM-2) of mucin as calculated from Lowry protein and hexose contents. Residual SDS could be removed by extraction of the mucin preparations with chloroform/ methanol (2: 1). Isolation of neutral oligosaccharide alditols from pronase-stable, high-molecular-mass glycopeptides The glycopeptide fraction (500 mg) was treated with 50 ml 0.05 M NaOH/1 M NaBH, (18 h at 50 'C) to release oligosaccharides by reductive p-elimination, neutralized with 25% (by vol.) acetic acid in the cold and decationized on a column (2.5 x 10 cm) of Dowex 50WX8 (hydrogen form) by elution with 5 vol. water. After freeze-drying, the residue was depleted of borate by repeated evaporation under reduced pressure with methanol containing 1 % acetic acid. Neutral alditols were separated from acidic, sialic-acid-containing carbohydrates by fractionation on a column (2.5 x 25 cm) of DEAESephadex A25 (acetate form) using a stepwise elution with two column volumes of 0.01 M (fraction N), 0.1 M (fraction Al), 0.25 M (fraction A,) and 0.5 M (fraction A3) pyridine/ acetate, pH 5.0. The fractions were freeze-dried, depleted of residual pyridine on Sephadex G25 (yields: 59 mg fraction N, 40 mg fraction Al, 14 mg fraction A2, 33.5 mg fraction A3) and neutral alditols in fraction N were subjected to further

529

I 1

Capillary GC/MS of partially methylated alditol acetates was performed by separation on a 15-m capillary column wallcoated with SE 54, heated from 100°C to 300°C at lO"C/ min and identification of the sugar derivatives by single-ion monitoring using dwell times of 0.02 s on an MSD 5970.

9

Electron-impact mass spectrometry of methylated oligosaccharides

I

0

w)

20

30

LO

time (min)

Fig. 1. HPLC of neutral glycan alditols from human amniotic mucins. Samples (1 mg) were chromatographed on a column (4.6 x 250 mm) of aminopropyl silica (Serva, Heidelberg, FRG) using gradient elution with acetonitrile/water (7: 3 to 1 :4) during 100 min. Eluting carbohydrates were registered spectrophotometrically at 192 nm. Peak numbers refer to the fractions N1 -N9 which were analyzed in this

study.

fractionation by HPLC (Fig. 1). HPLC was carried out on a Beckman system (model IIOA) using a Uvidec-100-111 variable-wavelength detector at 192 nm and a Shimadzu C-R1B computing integrator. Samples (1 -2 mg) were run on columns (4.6 x 250 mm) of silica bonded with aminopropylsilyl groups (Serva, Heidelberg, FRG) in a gradient of acetonitrile/water from 7: 3 to 1 :4 (duration 100 min) at a flow rate of 3 ml/min (Fig. 1). Carbohydrate-containing fractions were collected, freed from organic solvent by vacuum evaporation at 40°C and freeze-dried (yields from 10 mg fraction N: 1.1 mgN1,0.7mgN3,0.5mgN4, 1.0mgN5,0.6mgN6, 1.0 mg N7, 1.8 mg N9). Composition analyses Monosaccharide compositions of carbohydrate samples (10 pg) or glycoproteins (100 pg) were analyzed after methanolysis (0.65 M methanolic HC1) for 16 h at 70°C and reN-acetylation in pyridine (500 pl), methanol (50 pl), acetic anhydride (50 pl) for 15 min at ambient temperature. 1-0Methyl glycosides were trimethylsilylated using a mixture of N-methyl-N-trimethylsilyltrifluoroacetamideand trifluoroacetic acid (1O:l). The derivatives formed within 10 min at 70°C were analyzed on a fused silica capillary colum wallcoated with RSL 300 and heated in a gradient from 100°C to 130°C (16"C/min), followed by a gradient from 130°C to 260°C (4"Cjmin). Sulfate contents of mucin and alditol samples were assayed according to Silvestri et al. [12]. Methylation and methylation analysis Oligosaccharide alditols were methylated according to Hakomori's procedure and the partially methylated alditol acetates were prepared as described in [13]. Alditol samples (50- 100 pg) were methylated under dry argon in dimethylsulfoxide (50 pl) using sodium sulfinylcarbanion (50 pl) and methyl iodide (50 11) with short intervals of sonication during the solubilization and reaction steps. The methylated carbohydrates were extracted with chloroform/water, dried under a stream of nitrogen and subjected to the structural analyses.

Gas chromatography/mass spectrometry (GC/MS) of permethylated mono- to tetrasaccharide alditols was performed on a Hewlett Packard 5890 gas chromatograph coupled to a Hewlett Packard MSD 5970 quadrupol mass spectrometer. The samples were solubilized in chloroform and injected onto a capillary column wall-coated with SE54 (17 m), which was heated from 200°C to 320°C at 10"C/min. Mass spectra were recorded at 70 eV in cyclic scans from m/z 100 to 800. The temperatures of the interface and the ion source were set at 300°C and 250"C, respectively. Elution of methylated carbohydrates was also registered by single-ion monitoring of structurally relevant ion traces at mjz 187 (219 -32), 189,260,276,393,464,480 and 521 using dwell times of 0.02 s. Fast-atom-bombardment mass spectrometry FAB-MS was performed on a VG analytical ZAB-HF reversed geometry mass spectrometer. Spectra of underivatized neoglycolipids were recorded in the negative, those of the peracetylated or permethylated alditols in the positive ion mode. Neoglycolipids (1 - 2 pg) were analyzed after TLC by cutting the bands as strips and placing the aluminium-backed silica gel on a standard stainless steel target. A matrix of thioglycerol was used and solubilization of the neoglycolipids was aided by 2 pl methanol. Analysis of peracetylated saccharide alditols was performed after solubilization of the sample in 1 pl methanol mixed with 2 pl thioglycerol on the target, which was bombarded with xenon atoms having a kinetic energy equivalent to 8.5 - 9.5 kV. Preparation of neoglycolipids Neoglycolipids were prepared according to Stoll et al. [ll]. Briefly, oligosaccharide alditols (10 pg) dried in conical (0.3 ml) micro-vials (Piercc) were treated with 4 p1 1.25 mg/ml sodium periodate in 40 mM imidazole pH 6 at O T . After a 5-min incubation in the dark, the oxidation was stopped by addition of excess butan-2,3-diol(4.4 pg) and prolongation of the reaction time for a further 40 min. Schiff base formation of oxidized alditols with dipalmitoylglycerophosphoethanolamine (78 pg in chloroform/methanol, 1 : 1 ; 78 11) for 2 h at 50°C was followed by reduction with sodium cyanoborohydride (40 bg in 4 1 1 ethanol) overnight at 50°C. The reaction mixture was dried down in a stream ofnitrogen, solubilized in chloroform/methanol (1 :1) and applied onto high-performance thin-layer plates (silica 60, Merck, Darmstadt). The plate was successively developed with chloroform/methanol (1 : 1) and chloroform/methanol/water (1 30: 50:9) and stained with orcinol reagent. Exoglycosidase treatments Oligosaccharide alditols were solubilized in 50 mM sodium acetate pH 5.8 and treated with 0.1 units of the following exoglycosidases: cc-fucosidase from bovine kidney (Bochrin-

530 The highly glycosylated mucin fragments (T domains) obtained after extensive proteolytic digestion with pronase P were shown to be identical in size for both mucin species and to exhibit apparent molecular masses of approximately 500 kDa in SDS/PAGE (Fig. 2). Immunochemical analyses using a panel of carbohydrate-specific monoclonal antibodies revealed striking differences in the staining patterns of both mucin species (Table 1). While FM-1 expresses A, B and H blood group antigens and a broad spectrum of blood-grouprelated antigens, the only prominent carbohydrate antigens on FM-2 are represented by the sialylated derivatives of Lex and Lea antigens.

HPLC fractions N1 and N3 Fig. 2. SDS/PAGE of mucins and mucin-derived glycopeptides from human amniotic fluid. Glycoproteins (100 pg) were separated by clcctrophoresis in a 3 12% polyacrylamide gel (1.5 mm thick) in the prcsence of 0.1 YOSDS at 45 mA. Gels were fixed and stained with periodic acid/Schiff reagent. Lane 1 , pronasc-stable glycopeptides derived from amniotic mucins; lane 2, mucins from amniotic nuid isolatcd by phenol extraction followed by gel exclusion chromatography on Sephacryl S300. ~

ger, Mannheim, FRG), @-N-acetylglucosaminidasefrom jack beans (Sigma, Munich, FRG), p-galactosidase and a-galactosidase from Esckerichia coli (Sigma, Munich, FRG). The samples (100 pl) were incubated at 37°C under a layer of toluene for 24 h, heated for 3 min at 90°C and analyzed by HPLC after addition of 1 vol. acetonitrile and centrifugation.

CrO, oxidation of glycan alditols Approximately 20 pg glycan alditols, with 20 pg perseitol as internal standard, were acetylated with 0.2 ml pyridine/ acetic anhydride (1 : 1, by vol.) at ambient temperature for 18 h. Aftcr evaporation of the reaction mixture in a stream of nitrogen, half of the sample was oxidized in 0.2 ml acetic acid containing 20 mg C r 0 3 for 1 h at 50°C. The oxidized and the untreated sample were worked-up in parallel by repeated extraction with chloroform/water (1 :3 , by vol.). The dry residue of the combined organic phases was methanolyzed as described for the analysis of monosaccharide composition.

RESULTS Mucins in human amniotic fluid were enriched by phenol extraction followed by gel-exclusion chromatography on Sephacryl S-300 [4]. On analysis by SDSjPAGE in gradient gels (3 - 12%), two distinct molecular species, FM-1 and FM2 with apparent molecular masses of 700 and 570 kDa, respectively, could be identified after periodic acid/Schiff staining (Fig. 2). FM-1 and FM-2 were isolated by preparative SDS/ PAGE to apparent homogeneity. The carbohydrate content of FM-1 and FM-2 ranged between 70% (FM-2) and 80% (FM-1) by mass. The carbohydrate compositions of the mucin species were NeuAc, Fuc, Man, Gal, GalNAc, GlcNAc in the molar proportions 1.3:2.3:0.5:4.1:2.0:3.3 (FM-I) or 1.5:1.8:1.1 :4.0:1.0:2.9 (FM-2). On the basis of a 10-nmol detection limit, no sulfate could be detected in the mucin preparations (100 pg), in pronase-stable glycopeptides (1 00 pg) or in the fraction of neutral glycan alditols (50 pg).

On GC analysis of the trimethylsilylated alditol in fraction N1 one major peak was obtained which co-chromatographed with authentic GalNAc-01. The identity of N1-alditol with GalNAc-ol was further corroborated by FAB-MS analysis of the peracetylated alditol in the positive-ion mode (pseudomolecular ion at m/z 434; Table 2). Fraction N2 in the elution profile obtained on HPLC (Fig. 1) did not contain carbohydrate and was not subjected to further structural analyses. According to FAB-MS analysis of the permethylated or peracetylated alditol in fraction N3 (Table 2), the component carbohydrate corresponded to a disaccharide alditol composed of Hex (1) and HexNAc-ol (1) (pseudo-molecular ions at mjz 722 or 512). GC/MS analysis of the permethylated compound in fraction N3 revealed intense primary ions from the non-reducing or reducing ends at m / z 219 (Hex+) or 276 (HexNAc-ol'), respectively, and ions resulting from fragmentation of the terminal alditol residue (m/z378,422,466), which indicate a substitution of the HexNAc-ol in position C-3. The pattern of glycosidic substitution was confirmed by methylation analysis resulting in the formation of 1,5-di-O-acetylaled galactitol and 3-mono-0-acetylated N-acetylgalactosaminitol (Table 3) and by FAB-MS analysis in the negative-ion mode of the alditol-derived neoglycolipids (Table 4), which yielded a pseudo-molecular ion at m/z 997 (corresponding to oligosaccharide fragment Gal-3-OX) [l11. /?-Galactosidase and agalactosidase from E. coli were unable to cleave the galactosyl residue (Table 5). According to previous unpublished evidence the /?-specificenzyme does not attack the 8-galactosyl residue linked to position 3 of reduced N-acetylgalactosamine (see also resistance of the alditol in fraction N6). Anomeric configurations of galactosyl residues were determined, accordingly, by C r 0 3 oxidation (see below). The results can be summarized in the proposed structure for the alditol in fraction N3: Galp(1-3)GalNAc-01.

HPLC fraction N4 The oligosaccaride alditol in fraction N 4 represents a trisaccharide composed of dHex (l), Hex (1) and HexNAc-ol (1) as judged from the pseudo-molecular ion in FAB-MS of the permethylated or peracetylated alditol (Table 2). The EIMS obtained on GC/MS analysis of the permethylated compound was indicative of the linear sequence dHex-HexHexNAc-ol by exhibiting strong signals at mjz 189 (dHex '), 393 (dHex-Hex') and 276 (HexNAc-01'). Methylation analysis of N4 oligosaccharide revealed the formation of 1,5-di0-acetylated fucitol, 1,2,5-tri-O-acetylated galactitol and 3mono-0-acetylated N-acetylgalactosaminitol (Table 3). Substitution of the terminal GalNAc-ol in position C-3 was con-

53 1 Table 1. Carbohydrate epitopes on much species FM-1 and FM-2 from human amniotic fluid. Meconium was collected from a single blood group 0 individual, while mucins from amniotic fluid were from pooled samples. n.d. = not determined. Carbohydrate epitope

Antibody

Binding activity on

FM-1 Blood group untigens A-trisaccharide B-trisaccharide H2-trisaccharide Leatrisaccharide Lebtetrasaccharide LeYrisaccharide Leytetrasaccharide Blood group neoantigens Sialyl Lea

A005 BOO6 A46- RIB10 WOZ LebOl LeuMl 12-4LE 19-9

Sialyl Le"

{

Blood group precursor antigens (corc-type antigcns) T-disaccharide Sialyl-Tn disaccharide

Sialyl-6-T-trisaccharide

CSLEXl AM-3

~~~~

~

Pseudo-molecular ion M + H for alditols after acetylation or methylation

N1 N3 N4 N5

434 722 952 1009

N6 N7 N9

1239 1009 1239 1297

n.d 512 686 751 931 757 931 961

~

~

meconium glycoproteins

(+) -

-

-

+-

+++ ++ ++ n.d. ++++ +++ +++ ++++

-

(+I

+++ ++++ ++++

-

49H8 812.3 SP-21

Table 2. Pseudo-molecularions of peracetylated neutral mono- to tetrasaccharidc alditols registered by positive-ion fast-atom-bombardment mass spectrometry. n. d., not determined.

HPLC fraction

+++ +++ + (+) + + + ++ ++ ++

FM-2

~

+++ +++ Table 3. Methylation analysis of neutral mono-to tetrasaccharide alditols. Partially methylated alditol acetates

Proposed monosaccharide composition

HexNAc-ol HexHexN Ac-ol dHexHexHexNAc-ol Hex HexNAcHexNAc-ol d HexHexHexN AcHexNAc-ol HexHexNAcHexNAc-ol dHcxHexHexNAcHcxN Ac-ol Hex HexNAcHexNAc-ol

firmed also by the pseudo-molecular ion M-H in negative-ion FAB-MS of the alditol-derived neoglycolipid (Table 4). On treatment of the N4 alditol with a-fucosidase from bovine kidncy a complete conversion into N3-alditol was registered on HPLC (Table 5). In conjunction with results from C r 0 3 oxidation, the following structure can be assigned to the oligosaccharide in fraction N4: Fuca(l-2)Galp(l-3)GalNAc-01.

HPLC fraction N5 According to the pseudo-molecular ion in positive-ion FAB-MS of the permethylated or peracetylated alditols in fraction N5 the major component is identical to a trisaccharide alditol composed of Hex (l), HexNAc (1) and HexNAc-ol(1) (Table 2). The admixture of another major component became evident by the pseudo-molecular ions M + H at 1239 (peracetylylated derivative) or at 931 (permethylated derivative) indicating a tetrasaccharide with the composition dHex (I), Hex (l), HexNAc (1) and HexNAc-ol (1). The relative proportions of fucose-containing glycan alditols in fractions

HPLC fraction N3 N4 N5 N6 N 7 N9

Fucitol I ,5-di-O-acetyI Galactitol 1,5-di-O-acetyl 1,2,5-tri-O-acetyl N-Acet ylglucosaminitol 1,5-di-O-acetyl 1,3,5-tri-O-acetyl I ,4,5-tri-O-acetyl 1,3,4,5-tetra-O-acetyI

N - Acet ylgalactosamini to1 3-mono-O-acet yl 6-mono-0-acetyl 3,6-di-O-acetyl

- + + + - + + + + - - _ +- + - - + _ _ _ _

+ + + +_ _+ _+ _- +_ _ - _ + -

-

+ + -_ +

N5 and N7 varied with different batches of pronase-stable glycopeptides. Two major isomeric non-fucosylated oligosaccharides in this fraction could be separated after methykation by GC and identified by EI-MS with the trisaccharide sequences HexHexNAc-HexNAc-ol both yielding ions from the 'reducing' terminus at m / z 276 and 521 (Fig. 3). The substitution patterns of the isomeric trisaccharides were partially deduced from the EI-MS: compound N5a gave rise to the formation of ion series from the non-reducing terminus at m/z 187 (Hex+-32), 464 (Hex-HexNAc+) and 228 (Hex-HexNAc+-236)indicating the sequence Hexl -3HexNAc-HexNAc-01, while compound N5b yielded ion series at m/z 219 (Hex'), 187 (Hext-32), 464 (Hex-HexNAc') and 432 (Hex-HexNAc+-32) indicating the sequence Hexl -4HexNAc-HexNAc-01 (Fig. 3). The substitution of the terminal GalNAc-ol residue was derived from results of a methylation analysis (Table 3), FAB-

532 Table 4. Pseudo-molecular ions of underivatized neoglycolipids derived from neutral mono- to tetrasaccharide alditols registered by negativeion fast-atom-bombardmentmass spectrometry. HPLC fraction

Pseudomolecular ion M-H

Corresponding oligosaccharide fragments obtained from the C3 (3-OX) or C6 (6-OY) of tcrminal GalNAc-ol

A

loool 1

looo/

1

z w

Da 991 1143 1199 1345 997 937 1199 1345 1099 997

N3 N4 N5 N6 N7 N9

I-

5 600

Gal-3-OX Fuc,Gal-3-OX Gal,GlcNAc-3-OX Fuc,Gal, GlcNAc-3-OX Gal-3-OX GlcNAc-6-OY Gal,GlcNAc-3-OX Fuc, Gal, GlcNAc-3-OX Gal,GlcNAc-6-OY Gal-3-OX

>

W

I

I

1345 I

I 330

vl

E 3

A

MASS PER CHARGE

0

"- 8o01

c

ion521

Fig. 4. Thin-layer chromatography/negative-ion fast-atom-bombardment mass spectrometry of underivatized neoglycolipids. Neoglycolipids were chromatographed on HPTLC plates (Silica 60, Merck, Darmstadt, FRG) in chloroform/methanol/water(1 30: 50: 9). (A) Ncoglycolipids derived from alditols in fraction N5. (B) Neoglycolipids derived from alditols in fraction N7.

bl

c

? L

3 U

5

.-0 12

11

10

13

time (min)

B a

464

.

129

187

.-2. c

.-

I.,

200

MASS PER CHARGE

i

300

k!!,

,

.,!,

400

,

,

,

,

'..

, , C

500

E

mass/ charge

Fig. 3. Gas-liquid chromatograpby/mass spectrometry of permethylated glycan alditols in fraction N5. (A) Elution of methylated carbohydrates was registered by single-ion monitoring of ion traces at m/z 521 corresponding to the fragment HexNAc-HexNAc-01' . (B) Mass spectra were recorded at 70 eV in cyclic scans over m/z 100- 800. The tcrnperatures of the interface and of the ion source were set at 300°C and 250'C, respectively.

MS of the alditol-derived neoglycolipids (Table 4) and fragmentation in EI-MS (Fig. 3). The mixture of both compounds in fraction N5 yielded 3-mono-0-acetylated N-acetylgalactos-

aminitol in methylation analysis (Table 3). Two isomeric neoglycolipids of identical carbohydrate compo>itions, HexHexNAc-3-OX, and with similar mobilities in TLC were formed on derivatization of N5 alditols (Table 4, Fig. 4.). jGalactosidase from E. coli was able to release the terminal galactosyl residues thus indicating their anomeric configurations. The data obtained, including those from C r 0 3 oxidation (see below), can be summarized in the structure models of two isomeric trisaccharide alditols: Gal/j( 1-3)GlcNAc[j(13)GalNAc-01 and Gala(l-4)GlcNAcj( 1-3)GalNAc-01. The presence of a fucose-containing tetrasaccharide alditol became evident also on analysis of alditol-derived neoglycolipids by TLC/MS (m/z 1345) suggesting the structure dHexHex-HexNAc-3-OX (Table 4, Fig. 4).

HPLC fraction N6 Fraction N6 contains a trisaccharide composed of Hex (l), HexNAc (1) and HexNAc-ol(1) as represented by the pseudomolecular ion at m/z 1009 on FAB-MS of the peracetylated alditol (Table 2). GC/MS analysis of the permethylated compound in fraction N6 gave rise to the formation of intense primary fragments at m / z 219 (Hex'), 260 (HexNAc' ) and 521 (HexNAc-HexNAc-01') from the non-reducing or from the reducing end of the carbohydrate sequence, respectively. A branched core sequence of the trisaccharide was further corroborated by methylation analysis revealing a substitution of GalNAc-ol in positions C3 and C6 (Table 3) and by TLC/ MS analysis of alditol-derived neoglycolipids (Table 4). Two derivatives, Gal-3-OX and GlcNAc-6-OY1could be identified by their pseudo-molecular ions M-H at m/z 997 and 937, respectively. The trisaccharide was cleaved by enzymatic hydrolysis with a-N-acetylglucosaminidase (Table 5 ) yielding a disaccharide alditol isographic with the HPLC fraction N3.

533 Table 5. Exoglycosidase-catalyzedhydrolysis of terminal sugar residues. The symbol (-) indicates that cleavage of B-galactosyl residues was Incomplete, as demonstrated by HPLC of the rcaction products. Fraction

Resistance to hydrolytical cleavage by exoglycosidases ~-~ a-fucosidase fl-galactosidase a-galactosidase

+

N3 N4 NS N6 N7 N9

-

+ + + +

+ + + + + +

+ + (-1 + + (-1

On the basis or the combined data and Cr03 oxidation studies, the following structure is postulated for N6-alditol:

+ + ++ +

Table 6. Core structures established for 0-linked oligosaccharides on mucin-type glycoproteins. Structure

Gal/I(l- 3 )

Designation

\ GalNAc-ol

/ GlcNAcp(1- 6)

fl-N-acetylglucosaniinidase

GalNAc

/

core 1

Galj( 1 - 3) GlcNAcB(1- 6)

\ GalNAc

HPLC fraction N7 The major peracetylated oligosaccharide alditol in fraction N7 yielded a pseudo-molecular ion at m/z 1239 in positiveion FAB-MS (Table 2) representing a tetrasaccharide composed of dHex (l),Hex (I), HexNAc (1) and HexNAc-ol (1). The permethylated derivative gave rise to the formation of ion series at m/z 189 (dHex+), 157 (dHex+-methanol), 219 (Hex') and 187 (Hex'-methanol) in GC/MS indicating two non-reducing monosaccharide termini and at mjz 276 pointing to an unbranched GalNAc-ol terminus. Mono-3-substitution of CalNAc-ol became evident on analysis of partially methylated alditol acetates (Table 3) and on FAB-MS of the alditolderived neoglycolipids (Fig. 4 and Table 4), which revealed a derivative identical with dHexHexHexNAc-3-OX. The corresponding defucosylated derivative indicated by the ion at m j z 1345 was not detected by FAB-MS of the peracetylated or permethylated N7-alditols suggesting its formation during neoglycolipid synthcsis. The trisaccharide sequence Gall4(Fucl-3)GlcNAc is postulated to form a partial structure of the tetrasaccaride alditol in accordance with GCjMS and FAB-MS data (fragment series at mlz 638+432), a 3,4-di-0substituted GlcNAc derivative in methylation analysis (Table 3) and the resistance of the oligosaccharide to enzymatic cleavage by cc-fucosidasefrom bovine kidney (Table 5). The intense secondary ion tnjz 432 in GC/MS and the absence of a corresponding ion at m / z 402 (both derived from the primary fragment at mjz 638 representing a trisaccharide ion composed of dHexHexHexNAc) indicate that a fucosyl residue (instead of a galactosyl residue) should be linked to position C3 of the penultimate GlcNAc [3]. The residue in C3 of GlcNAc is prefcrentially eliminated with concomitant formation of a resonance-stabilized secondary ion. With regard to the observed exoglycosidase resistance of N7-aldito1, it should be emphasized that a-fucosidases from mammalian sources have been reported not to cleave fucose a3/4-linked to the penultimate N-acetylglucosamine residue [14]. The following structure can, accordingly, be assigned to the N7-alditol (refer also to results of Cr03-oxidation):

core 2

/ Galj( 1 - 3) GalNAc

/

core 3

GlcNAcP(1- 3 ) GlcNAcB(1 - 6)

\ GalNAc

core 4

/ GlcNAc/I(I - 3) GalNAc

/

core 5

GalNAca(1- 3) GlcNAcfi(1- 6 )

without generally accepted desigwtion

\ GalNAc

GalB( 1- 4)

\

GlcNAcij(1- 3)GalNAc-ol .

/ Fuca( I - 3 )

HPLC fraction N9 Fraction N9 contains a tetrasaccharide alditol with the composition Hex (2), HexNAc (I), HexNAc-ol(1) as revealed by the pseudo-molecular ion at m/z 1297 (Table 2). Due to the size and polarity of the compound, the permethylated N9alditol did not pass through the GC column under the conditions used. Sequence analysis was performed, accordingly, by direct-probe FAB-MS in the positive-ion mode revealing major signals at m/z 464 (Hex-HexNAc') and 432 (HexHexNAc+-MeOH) suggesting the disaccharide sequence Gal( 1-4)GlcNAc. The substitution pattern of the core GalNAc was established by two lines of evidence. On methylation analysis, a single GalNAc derivative was formed, which was identified

534 by GC/MS as 3,6-di-O-acetylated N-acetylgalactosaminitol (Table 3). Two neoglycolipids were derived from the alditol by oxidative cleavage and coupling to dipalmitoylglycerophosphoethanolamine: Gal-3-OX and Gal-GlcNAc-6-OY represented at rn/t 997 or 1099 on TLC/MS, respectively (Table 4). /l-Galactosidase from E. cofi cleaved one of the galactosyl residues and converted the N9-alditol into a trisaccharide isographic with N6 (Table 5). The combined use of figalactosidase ( E . coli) and b-N-acetylglucosaminidase (jack beans) resulted in the formation of N3-alditol. C r 0 3 oxidation did not reveal any indication for the presence of a-linked galactose in fraction N9. Summarizing the data from FAB-MS of the peracetylated compound, from methylation analysis, TLC/MS of the neoglycolipids and from C r 0 3 oxidation, the following structure can be assigned to the N9-alditol: Galfl(I

~

3)

\ GalNAc-ol .

/

Galfl(1 - 4)GlcNAcP( 1 - 6)

Anomeric configurations of glycosidic bonds While fucose in fractions N4, N5 and N7 was stable to oxidation by C r 0 3 (more than 80% retained), galactose and N-acetylglucosamine in fractions N3 - N9 were oxidized partially (less than 30% of galactose retained) or quantitatively (N-acetylglucosamine). Accordingly, fucose is presumably aglycosidically linked to the adjacent sugar in N4, N5 and N7, while galactose and N-acetylglucosamine should have a ficonfiguration.

DISCUSSION The amniotic cavity is filled with a clear, moderately viscous liquid, which is produced by the trophoblast-derived amniotic epithelia. Starting with the 5th month of gestation, the fetus swallows up to 400 ml amniotic fluid/day and in this way (via resorption) rapidly exchanges the aqueous portion of the fluid with the maternal circulation. During the final weeks of gestation the fetus excretes its low-concentrated urine into the fluid, while the contents of its intestinal tract, on the other hand, is released only under pathological conditions or under conditions of extreme psychical stress during birth. Macromolecular solutes of amniotic fluid like proteins or glycoproteins are, according to its origin, secretory products of the fetus or extra-fetal, but non-maternal epithelia. This explains why A, B, 0 blood group expression on amniotic mucins is strictly dependent on the blood group status of the fetus [2]. Moreover, we have shown previously by chemical and immunochemical analysis that mucins in amniotic fluid carry high proportions of sialylated fucosides belonging to the family of oncofetal carbohydrate structures [3, 41. In this contribution we present chemical evidence for a relationship between amniotic mucins and meconium glycoproteins, which could be regarded as a mixture of intestinal secretory products and components of concentrated amniotic fluid. 0-linked oligosaccharides on mucus glycoproteins from uneconiun and from amniotic fluid exhibit structural similarities with regard to the core sequences on short-chain glycans [7]. With the exception of the rarely expressed cores 4 and 5 (Table 6), the pattern of core-type sequences on amniotic mucins resembles that on meconium glycoproteins by com-

prising cores 1,2 and 3 in equally high proportions. Several of the presented carbohydrate structures have been ubiquitously found, while others, in particular core 3 sequences (Table 6), show a more restricted distribution in human tissues, since they have been detected previously on mucins from human meconium [7], colon [lo] and from the respiratory tract of patients suffering from bronchiectasis [I 51 or cystic fibrosis [I 61. The mucin fraction of amniotic fluid is composed of two chemically distinct species, which can be separated by electrophoresis and exhibit strikingly different glycosylation patterns, as revealed by immunochemical analysis. While FM1 (700 kDa) expresses high proportions of A, €3, H bloodgroup-active oligosaccharides, a completely different pattern of immunochemical reactivities was found for FM-2 (570 kDa), which is characterized by the predominant expression of sialylated fucosides (sialyl Lea, sialyl Le')). It remains to be established whether these immunochemically distinct mucins represent post-translational variants of the same gene product or whether they originate from different epithelia (fetal urothelium, amniotic epithelium) and could exhibit different peptide cores. Preliminary immunochemical data (unpublished results) suggest that the peptide portions of amniotic mucins display some sequence similarity with the polymorphic epithelial mucin (MUC1) previously described as an organ-characteristic much of the mammary gland and their secretions [17], but later detected also in the pancreas and urothelium. Even higher proportions of the polymorphic-epithelial-mucin-specificPDTRP sequence were found on chemically deglycosylated meconium glycoproteins (unpublished results). Despite this, the relationship of amniotic mucins to meconium glycoproteins remains obscure, although chemical analyses revealed some striking similarities with regard to the carbohydrate portions [3, 4, 6-81. On the other hand, meconium glycoproteins are distinguished from amniotic mucins by the pronounced expression of epitopes belonging to a family of sialyl-Tn-related antigens. Accordingly, the structural element NeuAccr(2-6)GalNAc is abundant in the glycan portion of meconium glycoproteins [8]. Further studies on the peptide portions and on the sitespecific glycan synthesis of fetal mucins will be needed for the elucidation of their glycosylation status and for a better understanding of differentiation-dependent shifts in the biosynthesis of mucin glycans. This investigation was supported by the Deutsche fiorschungsgemeinschuft grant Uh8/14-3. The authors gratefully acknowledge the skilful technical assistance of Mrs. E. JanRen. They also thank Prof. Dr. A. Bolte (University Clinic of Gynecology and Obstetrics, Cologne) for providing the samples of human amniotic fluid and Dr. W. Schanzer for his kind support during the GC/MS analyses.

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